The central objectives of this proposal are concerned with basic issues in the fields of molecular biology and endocrinology: we propose to investigate the events, mechanisms, and membrane structures responsible for pre-hormone processing, i.e. binding to and translocation across the rough endoplasmic reticulum (RER), and cleavage of the leader sequence from the remaining nascent polypeptides. Most, if not all, polypeptide hormones and other secreted proteins are biosynthesized as precursors that transiently contain NH2-terminal extensions termed leader, or signal, sequences. It is believed that these sequences have a specialized and essential role in intracellular transport and metabolism of secreted proteins and that their interaction with a membrane-associated processing apparatus determines whether a nascent protein remains intracellular or is secreted. To study the processing events and structures at the molecular level, we propose to use chemically synthesized pre-proparathyroid hormone (Pre-ProPTH), or fragments and analogues of the precursor-specific region of the molecule, and cell-free translational and processing systems. Structure/activity relations of the leader sequence will be studied: the minimum sequence necessary for binding, translocation, and pre-hormone cleavage will each be determined, and critical structural features for bioactivity will be elucidated. Photoaffinity labeling of elements of the RER processing apparatus will be performed, permitting subsequent harvesting and characterization of these membrane components. Antisera to these membrane structures and to the precursor region of Pre-ProPTH will be generated to undertake immunocytolocalization studies and determine whether the RER processing apparatus is a universal constituent of eukaryotic cells. These investigations, based on a direct approach through chemical peptide synthesis and in vitro biological studies, should provide fundamental insight into the normal physiology of hormonal and protein secretion and identify the critical sites and steps at which there might be dysfunction that could lead to deficiencies or alterations in hormonal secretion or metabolism.